Nitrosoureas including BCNU, CCNU, methyl-CCNU and chlorozotocin are of clinical value in the treatment of a broad spectrum of neoplasms, including CNS diseases, leukemia, Hodgkin's disease and others. Present evidence favors the nucleic acids as one of the principal cell target sites. While much empirical structure-antileukemic activity data exists, the molecular mechanism of action is not understood which is preventing rational structure modification to enhance antitumor properties. In the next phase of our work the first objective is to clarify certain aspects of the decomposition mechanism using specifically deuterated nitrosoureas. Then several suggested plausible breakdown products of the nitrosoureas will be synthesized and their chemical interaction, together with those of the parent nitrosoureas, with DNA will be examined. Now that we have successfully isolated 2-alkylimino-oxazolidines as important intermediates from BCNU, CCNU, and MeCCNU their relative contribution to the action of each of these agents will be assessed. These studies will employ radioactivity incorporation, gel electrophoresis and sensitive and versatile ethidium fluorescence assays which have proven utility in the study of the mechanisms of action of several antitumor agents. Attempts will be made to isolate by selective enzymatic degradation and to identify the cross-linked site in DNA produced by nitrosoureas. Efforts will be made to correlate extent, type and rate of chemical lesions in DNA with the antitumor properties of nitrosoureas as has been done successfully with other agents. An understanding of the chemical basis of the mode of action will be invaluable in providing data for rational structural design to optimize antileukemic properties. Thus based on our findings the synthesis and in vivo testing of new nitrosoureas we commmenced in the first phase will be extended.